This project is designed to develop a novel mouse model of human mitochondrial disease. Genetic engineering and molecular biological techniques will be utilized to create heteroplasmic "transmitochondrial" mice harboring a mutant mitochondrial genome. This model and the procedures for mitochondrial transfer will be of considerable importance toward our understanding of a specific mitochondrial mutation, as well as leading to the development of novel strategies and therapies for human metabolic diseases influenced by aberrations in mitochondrial function or mutation. In pilot experiments, the ability to create transmitochondrial mouse models that transmit the heteroplasmic state to offspring in maternal lineages was identified. With the advent of gene transfer technologies and PCR-based procedures, this project will target the development of technology to establish a mouse model harboring a directed mitochondria DNA (mtDNA) deletion. Specific Aims include: (1) development and optimization of mitochondria transfection procedures, (2) transfer of transfected mitochondria (or cells) into mouse ova to produce heteroplasmic transmitochondrial mice that will recapitulate a deletion associated with human mtDNA-based disease - for which no animal model exists for study, and (3) characterization of transmitochondrial mouse lineages created over the course of this project. Production of heteroplasmic transmitochondrial deletion mutants will be a critical first step, facilitating the study of mitochondrial function and disease progression. Initially, this model will serve to explore disease pathogenesis and mitochondrial dynamics in an in vivo system. Ultimately, transmitochondrial mouse models will be used to explore the role of the mitochondrial genome in human metabolic disease processes and in the development of novel human gene therapies.